Stimulation of numerous G-protein coupled receptors leads to elevation of intracellular concentrations of cAMP, which subsequently activates the cAMP-dependent protein kinase (PKA) pathway. Specificity of the PKA signaling module is determined by a sophisticated subcellular targeting network, which directs the spatio-temporal activation of the kinase. This specific compartmentalization mechanism occurs through high-affinity interaction of the PKA with A-kinase anchoring proteins (AKAPs), the role of which is to target the kinase to discrete subcellular microdomains. Recently, a peptide, designated "AKAPis", has been proposed to competitively inhibit PKA-AKAP interactions in vitro. We therefore sought to characterize a cell-permeable construct of AKAPis inhibitor, and use it as a tool to characterize the impact of PKA compartmentalization by AKAPs. Using insulin-secreting, INS-1, pancreatic beta cells, we show that TAT-AKAPis (at a micromolar range) dose-dependently disrupted a significant fraction of endogenous PKA-AKAP interactions. Immunoflurescence analysis also indicated that TAT-AKAPis affected significantly PKA subcellular localization. Furthermore, TAT-AKAPis markedly attenuated the glucagon-induced phosphorylations of p44/p42 MAPKs and CREB, which are downstream effectors of PKA. In parallel, TAT-AKAPis dose-dependently inhibited the glucagon-induced potentiation of insulin release. Therefore, AKAP-mediated subcellular compartmentalization of PKA represents a key mechanism for PKA-dependent phosphorylation events and potentiation of insulin secretion in intact pancreatic beta cells. More interestingly, our data highlight the effectiveness of CPP-mediated approach to monitoring in cellulo PKA-AKAP interactions and delineating PKA-dependent phosphorylation events underlying specific cellular responses.